Plant YTHDF proteins are direct effectors of antiviral immunity against an N6-methyladenosine-containing RNA virus.

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of a plant positive-strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6-methyladenosine (m6 A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521-B homology domain (YTH)-containing m6 A-binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m6 A-YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

Transcriptomic profiling reveals the complex interaction between a bipartite begomovirus and a cucurbitaceous host plant.

BACKGROUND: Begomoviruses are major constraint in the production of many crops. Upon infection, begomoviruses may substantially modulate plant biological processes. While how monopartite begomoviruses interact with their plant hosts has been investigated extensively, bipartite begomoviruses-plant interactions are understudied. Moreover, as one of the major groups of hosts, cucurbitaceous plants have been seldom examined in the interaction with begomoviruses. RESULTS: We profiled the zucchini transcriptomic changes induced by a bipartite begomovirus squash leaf curl China virus (SLCCNV). We identified 2275 differentially-expressed genes (DEGs), of which 1310 were upregulated and 965 were downregulated. KEGG enrichment analysis of the DEGs revealed that many pathways related to primary and secondary metabolisms were enriched. qRT-PCR verified the transcriptional changes of twelve selected DEGs induced by SLCCNV infection. Close examination revealed that the expression levels of all the DEGs of the pathway Photosynthesis were downregulated upon SLCCNV infection. Most DEGs in the pathway Plant-pathogen interaction were upregulated, including some positive regulators of plant defenses. Moreover, the majority of DEGs in the MAPK signaling pathway-plant were upregulated. CONCLUSION: Our findings indicates that SLCCNV actively interact with its cucurbitaceous plant host by suppressing the conversion of light energy to chemical energy and inducing immune responses. Our study not only provides new insights into the interactions between begomoviruses and host plants, but also adds to our knowledge on virus-plant interactions in general.

Transcriptional and hormonal profiling uncovers the interactions between plant developmental stages and RNA virus infection.

Arabidopsis thaliana is more susceptible to certain viruses during its later developmental stages. The differential responses and the mechanisms behind this development-dependent susceptibility to infection are still not fully understood. Here we explored the outcome of a viral infection at different host developmental stages by studying the response of A. thaliana to infection with turnip mosaic virus at three developmental stages: juvenile vegetative, bolting, and mature flowering plants. We found that infected plants at later stages downregulate cell wall biosynthetic genes and that this downregulation may be one factor facilitating viral spread and systemic infection. We also found that, despite being more susceptible to infection, infected mature flowering plants were more fertile (i.e. produce more viable seeds) than juvenile vegetative and bolting infected plants; that is, plants infected at the reproductive stage have greater fitness than plants infected at earlier developmental stages. Moreover, treatment of mature plants with salicylic acid increased resistance to infection at the cost of significantly reducing fertility. Together, these observations support a negative trade-off between viral susceptibility and plant fertility. Our findings point towards a development-dependent tolerance to infection.

Silencing of a Nicotiana benthamiana ascorbate oxidase gene reveals its involvement in resistance against cucumber mosaic virus.

MAIN CONCLUSION: Silencing of an ascorbate oxidase (AO) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV. A Nicotiana benthamiana ascorbate oxidase (NbAO) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana. The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO-silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was "occupied" by TRV. In contrast, in the NbAO-silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana.

A resilient bunch: stem cell antiviral immunity in plants.

Stem cells are vital for plant development and reproduction. The stem cells within shoot apical meristems are known to possess exceptionally effective antiviral defenses against pathogenic viruses which preclude their infection, yet how this is achieved remains poorly understood and scarcely investigated. In this Tansley Insight, we connect very recent experimental results with previous work to summarize the known molecular mechanisms determining stem cell antiviral immunity. More broadly, we attempt to define the viral features triggering immunity and the global consequences of virus infection in these essential cells. This brief article will highlight how these phenomena are fascinating, complex and often crucial for virus-host interactions, while emphasizing the potential for discovery in their investigation.

The role of reactive oxygen species in plant-virus interactions.

Reactive oxygen species (ROS) play a complex role in interactions between plant viruses and their host plants. They can both help the plant defend against viral infection and support viral infection and spread. This review explores the various roles of ROS in plant-virus interactions, focusing on their involvement in symptom development and the activation of plant defense mechanisms. The article discusses how ROS can directly inhibit viral infection, as well as how they can regulate antiviral mechanisms through various pathways involving miRNAs, virus-derived small interfering RNAs, viral proteins, and host proteins. Additionally, it examines how ROS can enhance plant resistance by interacting with hormonal pathways and external substances. The review also considers how ROS might promote viral infection and transmission, emphasizing their intricate role in plant-virus dynamics. These insights offer valuable guidance for future research, such as exploring the manipulation of ROS-related gene expression through genetic engineering, developing biopesticides, and adjusting environmental conditions to improve plant resistance to viruses. This framework can advance research in plant disease resistance, agricultural practices, and disease control.

Proteolytic Processing of Plant Proteins by Potyvirus NIa Proteases.

The NIa protease of potyviruses is a chymotrypsin-like cysteine protease related to the picornavirus 3C protease. It is also a multifunctional protein known to play multiple roles during virus infection. Picornavirus 3C proteases cleave hundreds of host proteins to facilitate virus infection. However, whether or not potyvirus NIa proteases cleave plant proteins has so far not been tested. Regular expression search using the cleavage site consensus sequence [EQN]xVxH[QE]/[SGTA] for the plum pox virus (PPV) protease identified 90 to 94 putative cleavage events in the proteomes of Prunus persica (a crop severely affected by PPV), Arabidopsis thaliana, and Nicotiana benthamiana (two experimental hosts). In vitro processing assays confirmed cleavage of six A. thaliana and five P. persica proteins by the PPV protease. These proteins were also cleaved in vitro by the protease of turnip mosaic virus (TuMV), which has a similar specificity. We confirmed in vivo cleavage of a transiently expressed tagged version of AtEML2, an EMSY-like protein belonging to a family of nuclear histone readers known to be involved in pathogen resistance. Cleavage of AtEML2 was efficient and was observed in plants that coexpressed the PPV or TuMV NIa proteases or in plants that were infected with TuMV. We also showed partial in vivo cleavage of AtDUF707, a membrane protein annotated as lysine ketoglutarate reductase trans-splicing protein. Although cleavage of the corresponding endogenous plant proteins remains to be confirmed, the results show that a plant virus protease can cleave host proteins during virus infection and highlight a new layer of plant-virus interactions. IMPORTANCE Viruses are highly adaptive and use multiple molecular mechanisms to highjack or modify the cellular resources to their advantage. They must also counteract or evade host defense responses. One well-characterized mechanism used by vertebrate viruses is the proteolytic cleavage of host proteins to inhibit the activities of these proteins and/or to produce cleaved protein fragments that are beneficial to the virus infection cycle. Even though almost half of the known plant viruses encode at least one protease, it was not known whether plant viruses employ this strategy. Using an in silico prediction approach and the well-characterized specificity of potyvirus NIa proteases, we were able to identify hundreds of putative cleavage sites in plant proteins, several of which were validated by downstream experiments. It can be anticipated that many other plant virus proteases also cleave host proteins and that the identification of these cleavage events will lead to novel antiviral strategies.

AtGSTU19 and AtGSTU24 as Moderators of the Response of Arabidopsis thaliana to Turnip mosaic virus.

Plants produce glutathione as a response to the intercellular redox state. Glutathione actively participates in the reactive oxygen species (ROS)-dependent signaling pathway, especially under biotic stress conditions. Most of the glutathione S-transferases (GSTs) are induced in cells during the defense response of plants not only through highly specific glutathione-binding abilities but also by participating in the signaling function. The tau class of GSTs has been reported to be induced as a response under stress conditions. Although several studies have focused on the role of the tau class of GSTs in plant-pathogen interactions, knowledge about their contribution to the response to virus inoculation is still inadequate. Therefore, in this study, the response of Atgstu19 and Atgstu24 knockout mutants to mechanical inoculation of Turnip mosaic virus (TuMV) was examined. The systemic infection of TuMV was more dynamically promoted in Atgstu19 mutants than in wild-type (Col-0) plants, suggesting the role of GSTU19 in TuMV resistance. However, Atgstu24 mutants displayed virus limitation and downregulation of the relative expression of TuMV capsid protein, accompanied rarely by TuMV particles only in vacuoles, and ultrastructural analyses of inoculated leaves revealed the lack of virus cytoplasmic inclusions. These findings indicated that Atgstu24 mutants displayed a resistance-like reaction to TuMV, suggesting that GSTU24 may suppress the plant resistance. In addition, these findings confirmed that GSTU1 and GSTU24 are induced and contribute to the susceptible reaction to TuMV in the Atgstu19-TuMV interaction. However, the upregulation of GSTU19 and GSTU13 highly correlated with virus limitation in the resistance-like reaction in the Atgstu24-TuMV interaction. Furthermore, the highly dynamic upregulation of GST and glutathione reductase (GR) activities resulted in significant induction (between 1 and 14 days post inoculation [dpi]) of the total glutathione pool (GSH + GSSG) in response to TuMV, which was accompanied by the distribution of active glutathione in plant cells. On the contrary, in Atgstu19, which is susceptible to TuMV interaction, upregulation of GST and GR activity only up to 7 dpi symptom development was reported, which resulted in the induction of the total glutathione pool between 1 and 3 dpi. These observations indicated that GSTU19 and GSTU24 are important factors in modulating the response to TuMV in Arabidopsis thaliana. Moreover, it was clear that glutathione is an important component of the regulatory network in resistance and susceptible response of A. thaliana to TuMV. These results help achieve a better understanding of the mechanisms regulating the Arabidopsis-TuMV pathosystem.

Glutathione Modulation in PVYNTN Susceptible and Resistant Potato Plant Interactions.

Glutathione is a metabolite that plays an important role in plant response to biotic stress through its ability to remove reactive oxygen species, thereby limiting the degree of potential oxidative damage. It can couple changes in the intracellular redox state to the development, especially the defense responses, of plants. Several studies have focused on measuring glutathione levels in virus infected plants, but have not provided complete information. Therefore, we analyzed, for the first time, the content of glutathione as well as its ultrastructural distribution related to susceptible and hypersensitive potato-Potato virus Y NTN (PVYNTN) interaction, with an aim of providing new insight into interactive responses to PVYNTN stress. Our findings reported that the inoculation of PVYNTN caused a dynamic increase in the content of glutathione, not only in resistance but also in susceptible reaction, especially at the first steps of plant-virus interaction. Moreover, the increase in hypersensitive response was much more dynamic, and accompanied by a significant reduction in the content of PVYNTN. By contrast, in susceptible potato Irys, the content of glutathione decreased between 7 and 21 days after virus inoculation, which led to a significant increase in PVYNTN concentration. Additionally, our findings clearly indicated the steady induction of two selected potato glutathione S-transferase StGSTF1 and StGSTF2 genes after PVYNTN inoculation, regardless of the interaction type. However, the relative expression level of StGSTF1 did not significantly differ between resistant and susceptible plants, whereas the relative expression levels of StGSTF2 differed between susceptible and resistant reactions. Therefore, we proposed that StGSTF2 can act as a marker of the type of response to PVYNTN. Our observations indicated that glutathione is an important component of signaling as well as the regulatory network in the PVYNTN-potato pathosystem. In resistance responses to PVYNTN, this metabolite activates plant defenses by reducing potential damage to the host plant cell, causing a reduction in virus concentration, while it can also be involved in the development of PVYNTN elicited symptoms, as well as limiting oxidative stress, leading to systemic infection in susceptible potato plants.

Viral Fitness Determines the Magnitude of Transcriptomic and Epigenomic Reprograming of Defense Responses in Plants.

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.

Host population structure for tolerance determines the evolution of plant-virus interactions.

Heterogeneity for plant defences determines both the capacity of host populations to buffer the effect of infection and the pathogen´s fitness. However, little information is known on how host population structure for tolerance, a major plant defence, impacts the evolution of plant-pathogen interactions. By performing 10 serial passages of Turnip mosaic virus (TuMV) in Arabidopsis thaliana populations with varying proportion of tolerant genotypes simulating different structures for this trait, we analysed how host heterogeneity for this defence shapes the evolution of both virus multiplication, the effect of infection on plant fecundity and mortality, and plant tolerance and resistance. Results indicated that a higher proportion of tolerant genotypes in the host population promotes virus multiplication and reduces the effect of infection on plant mortality, but not on plant fecundity. These changes resulted in more effective plant tolerance to virus infection. Conversely, a lower proportion of tolerant genotypes reduced virus multiplication, boosting plant resistance. Our work for the first time provides evidence of the main role of host population structure for tolerance on pathogen evolution and on the subsequent feedback loops on plant defences.

Gene Expression Analysis of Induced Plum pox virus (Sharka) Resistance in Peach (Prunus persica) by Almond (P. dulcis) Grafting.

No natural sources of resistance to Plum pox virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a "Garrigues" almond scion onto "GF305" peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation. Furthermore, grafting a "Garrigues" scion onto the "GF305" rootstock has been shown to completely prevent virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting "Garrigues" almond onto healthy "GF305" peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by "Garrigues" almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the "Garrigues"-induced response, since its expression is much higher in "Garrigues" than in "GF305". We also discuss the potential relevance of the following in PPV infection and "Garrigues"-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.

Positive Selection in the Chloroplastic ATP-Synthase ß-Subunit and Its Relation to Virulence Factors.

The most paradigmatic examples of molecular evolution under positive selection involve genes related to the immune system. Recently, different chloroplastic factors have been shown to be important for plant defenses, among them, the α- and ß-subunits of the ATP synthase. The ß-subunit has been reported to interact with several viral proteins while both proteins have been implicated with sensitivity to tentoxin, a phytotoxin produced by the widespread fungus Alternaria alternata. Given the relation of both protein to virulence factors, we studied whether these proteins are evolving under positive selection. To this end, we used the dN/dS ratio to examine possible sites under positive selection in several Angiosperm clades. After examining 79 plant genera and 1232 species, we found three times more sites under pervasive diversifying selection in the N-terminal region of the ß-subunit compared to the α-subunit, supporting previous results which identified this region as responsible for interacting with viral proteins. Moreover, we found the site 83 of ß-subunit under positive selection in several plant genera, a site clearly related to the sensitivity to tentoxin according to biochemistry assays, which possibly reflects the selective pressure of the non-host specific tentoxin across various Angiosperm clades.

Defence priming in tomato by the green leaf volatile (Z)-3-hexenol reduces whitefly transmission of a plant virus.

Green leaf volatiles (GLVs) can induce defence priming, that is, can enable plants to respond faster or more strongly to future stress. The effects of priming by GLVs on defence against insect herbivores and pathogens have been investigated, but little is known about the potential of GLVs to prime crops against virus transmission by vector insects. Here, we tested the hypothesis that exposure to the GLV Z-3-hexenol (Z-3-HOL) can prime tomato (Solanum lycopersicum) for an enhanced defence against subsequent Tomato yellow leaf curl virus (TYLCV) transmission by the whitefly Bemisia tabaci. Bioassays showed that Z-3-HOL priming reduced subsequent plant susceptibility to TYLCV transmission by whiteflies. Z-3-HOL treatment increased transcripts of jasmonic acid (JA) biosynthetic genes and increased whitefly-induced transcripts of salicylic acid (SA) biosynthetic genes in plants. Using chemical inducers, transgenics and mutants, we demonstrated that induction of JA reduced whitefly settling and successful whitefly inoculation, while induction of SA reduced TYLCV transmission by whiteflies. Defence gene transcripts and flavonoid levels were enhanced when whiteflies fed on Z-3-HOL-treated plants. Moreover, Z-3-HOL treatment reduced the negative impact of whitefly infestation on tomato growth. These findings suggest that Z-3-HOL priming may be a valuable tool for improving management of insect-transmitted plant viruses.

Respiratory Burst Oxidase Homologs RBOHD and RBOHF as Key Modulating Components of Response in Turnip Mosaic Virus-Arabidopsis thaliana (L.) Heyhn System.

Turnip mosaic virus (TuMV) is one of the most important plant viruses worldwide. It has a very wide host range infecting at least 318 species in over 43 families, such as Brassicaceae, Fabaceae, Asteraceae, or Chenopodiaceae from dicotyledons. Plant NADPH oxidases, the respiratory burst oxidase homologues (RBOHs), are a major source of reactive oxygen species (ROS) during plant-microbe interactions. The functions of RBOHs in different plant-pathogen interactions have been analyzed using knockout mutants, but little focus has been given to plant-virus responses. Therefore, in this work we tested the response after mechanical inoculation with TuMV in ArabidopsisrbohD and rbohF transposon knockout mutants and analyzed ultrastructural changes after TuMV inoculation. The development of the TuMV infection cycle was promoted in rbohD plants, suggesting that RbohD plays a role in the Arabidopsis resistance response to TuMV. rbohF and rbohD/F mutants display less TuMV accumulation and a lack of virus cytoplasmic inclusions were observed; these observations suggest that RbohF promotes viral replication and increases susceptibility to TuMV. rbohD/F displayed a reduction in H2O2 but enhanced resistance similarly to rbohF. This dominant effect of the rbohF mutation could indicate that RbohF acts as a susceptibility factor. Induction of hydrogen peroxide by TuMV was partially compromised in rbohD mutants whereas it was almost completely abolished in rbohD/F, indicating that these oxidases are responsible for most of the ROS produced in this interaction. The pattern of in situ H2O2 deposition after infection of the more resistant rbohF and rbohD/F genotypes suggests a putative role of these species on systemic signal transport. The ultrastructural localization and quantification of pathogenesis-related protein 1 (PR1) indicate that ROS produced by these oxidases also influence PR1 distribution in the TuMV-A.thaliana pathosystem. Our results revealed the highest activation of PR1 in rbohD and Col-0. Thus, our findings indicate a correlation between PR1 accumulation and susceptibility to TuMV. The specific localization of PR1 in the most resistant genotypes after TuMV inoculation may indicate a connection of PR1 induction with susceptibility, which may be characteristic for this pathosystem. Our results clearly indicate the importance of NADPH oxidases RbohD and RbohF in the regulation of the TuMV infection cycle in Arabidopsis. These findings may help provide a better understanding of the mechanisms modulating A.thaliana-TuMV interactions.

Contribution of Tomato torrado virus Vp26 coat protein subunit to systemic necrosis induction and virus infectivity in Solanum lycopersicum.

BACKGROUND: Tomato torrado virus (ToTV) infection manifests with burn-like symptoms on leaves, leaflets and upper stem parts of susceptible infected plants. The symptoms caused by ToTV may be considered as one of the most severe virus-induced forms of systemic necrosis, which spreads within the whole plant and leads to a lethal phenotype. However, to date there are no data revealing which viral genes encode for a specific pathogenicity determinant that triggers the plant necrotic response for any torradovirus. In this study we evaluated the influence of three coat protein subunits of ToTV: Vp23, Vp26 and Vp35, transiently expressed from a PVX-based vector, and checked their association with the induction of systemic necrosis in infected Solanum lycopersicum L. (cv. Beta Lux), a natural host of ToTV. METHODS: To estimate how ToTV coat protein subunits might contribute in plant response to virus infection we over-expressed the proteins from PVX-based vector in tomato and analyzed enzymatic activities related with plant defense response. By doing protein qualitative analysis performed by mass spectrometry we indicated the PR10 in protein fraction with induced ribonuclease activity. RESULTS: We observed that only the Vp26 enhanced PVX pathogenicity causing severe necrosis of the infected plant. Moreover, we indicated increased RNase and oxidative activities in plants infected with PVX-Vp26 chimeras only. Importantly, we suspected that this increased RNase activity is associated with increased accumulation of PR10 mRNA and products of its translation. CONCLUSIONS: On the basis of the obtained results, we indicated that Vp26 acts as the elicitor of hypersensitive response-like reactions of PVX-Vp26 manifesting with enhanced pathogenicity of the recombined PVX. This might be the first described suspected necrosis determinant of torradoviruses infecting tomatoes.

Assessment of the Efficacy and Mode of Action of Benzo(1,2,3)-Thiadiazole-7-Carbothioic Acid S-Methyl Ester (BTH) and Its Derivatives in Plant Protection Against Viral Disease.

Systemic acquired resistance (SAR) induction is one of the primary defence mechanisms of plants against a broad range of pathogens. It can be induced by infectious agents or by synthetic molecules, such as benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). SAR induction is associated with increases in salicylic acid (SA) accumulation and expression of defence marker genes (e.g., phenylalanine ammonia-lyase (PAL), the pathogenesis-related (PR) protein family, and non-expressor of PR genes (NPR1)). Various types of pathogens and pests induce plant responses by activating signalling pathways associated with SA, jasmonic acid (JA) and ethylene (ET). This work presents an analysis of the influence of BTH and its derivatives as resistance inducers in healthy and virus-infected plants by determining the expression levels of selected resistance markers associated with the SA, JA, and ET pathways. The phytotoxic effects of these compounds and their influence on the course of viral infection were also studied. Based on the results obtained, the best-performing BTH derivatives and their optimal concentration for plant performance were selected, and their mode of action was suggested. It was shown that application of BTH and its derivatives induces increased expression of marker genes of both the SA- and JA-mediated pathways.

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics.

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC⁻MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV⁻host interaction that might contribute to the symptom development.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.